Hematology reference control, as a liquid comprising single or multi-component blood cells or blood cell analogs, which is as detectable as blood, is used for routinely monitoring the accuracy and precision of hematology analyzers. Along with the continuing development of hematology analyzers, the accompanying controls should also solve the problems of increased parameters, for example, the development of three-part differential white blood cell control to five-part differential white blood cell control or controls with more parameters. Compared with three-part differential hematology analyzers, five-part differential hematology analyzers make more use of electrical, optical, chemical staining characteristics or the like of white blood cells to achieve five-part differential detection of white blood cells. As different types of five-part differential hematology analyzers vary in principles of detection and the corresponding accompanying white blood cell controls also rely on varying principles, emphasis is more placed on “tailor-making”. As is mentioned in U.S. Pat. No. 6,762,055, an accompanying control used in a certain type of hematology analyzer quite probably may not work efficiently in another type.
Attempts have been made to simulate five-part differential white blood cell controls using pollen, latex particles, various organic materials and fixed red blood cells. The methods of the prior art for preparing five-part differential white blood cell analogs can be summarized as follows: 1) treating animal red blood cells for simulation; and 2) treating mammal white blood cells (generally human white blood cells) for simulation.
When animal red blood cells are used for simulating five-part differential white blood cell components, the requirements for cell characteristics expand from the simple parameter of volume size to the characteristics such as conductivity, light scattering and cell chemical staining. Therefore, animal red blood cells should be subjected to proper treatment. For example, as is mentioned in U.S. Pat. Nos. 5,320,964 and 5,512,485, white blood cell analogs are comprised of red blood cells capable of simulating at least two kinds of physical properties of human white blood cells. Monocytes, lymphocytes and neutrophils are simulated by treating reptile red blood cells through a certain method as using a hypotonic solution containing an aldehyde fixing agent to change the hemoglobin content of the red blood cells, thereby changing their optical and electrical properties. In another example, human eosinophils are simulated using a hemoglobin denaturing solution to denature and precipitate the hemoglobin in red blood cells. U.S. Pat. No. 6,146,901 also describes to treat red blood cells with a mixed solution containing a polyhydric alcohol, a fixing agent and/or a non-ionic surfactant to obtain the desired white blood cell characteristics through changing the relative concentration of these components and their interaction time. These white blood cell analogs were finally mixed with soluble human red blood cells and stable platelets or platelet analogs to make up a multi-parameter whole blood control.
However, simulation of human white blood cells using animal red blood cells suffers from the disadvantage of limited simulation of individual characteristics of a certain type of white blood cell, as the animal red blood cells differ greatly from human white blood cells in morphology, membrane, cytoplasm granularity, nucleus and other inner structures. Moreover, where different types of five-part differential hematology analyzers vary in principles of detection, it is likely that a certain type of white blood cell particles simulated on a certain type of hematology analyzer might be represented as cell debris or other abnormalities on another type. Even though changing hemoglobin content and properties of red blood cells as described in the above-mentioned patents, it is not possible to completely simulate the various characteristics of different types of white blood cells. In particular, at the aspects of fluorescent binding, particle complexity and cell chemical staining etc., animal red blood cells hardly manifest the detection characteristics consistent with those of real white blood cells, which is a limitation to the application of the processing method. As for a five-part differential hematology analyzer, it is difficult and complicated to realize the simulation of five-part differential white blood cells using animal red blood cells, and compatibility is quite poor. Experimental results obtained by the present inventor also shows that white blood cell particles simulated by animal erythroblasts on a hematology analyzer based on the principle of multi-angle light scattering are too weak in cell clustering to obtain precise white blood cell differentiation. See the scattergram profiles in FIGS. 1 and 2. It can also be seen from the disclosed patents that animal red blood cells are not used for completely simulating human five-part differential white blood cells used in the quality control of five-part differential hematology analyzers.
Use of mammal white blood cells for preparation of five-part differential white blood cell analogs provides a better means to cope with complexity in reactions during the detection process of hematology analyzers varying in principles. On the whole, the methods in the prior art for treating white blood cells comprise the steps of hemolysis, fixation and washing, etc. For example, U.S. Pat. Nos. 6,406,915, 6,403,377, 6,399,388, 6,221,668, 6,200,500, 5,981,282, 5,731,205, 5,677,145, 5,672,474, 5,270,208, 5,262,327 describe to use the hemolytic agent Tris-ammonium chloride to completely lyse the red blood cells, and separate and then fix the human white blood cells. The white blood cell analogs were finally preserved in the preservation solution added with lipoprotein such that they approach the characteristics of real white blood cells. U.S. Pat. Nos. 6,762,055 and 6,514,763 also mainly concern the processes of removing red blood cells using mild chemical methods to retain intact white blood cells, then fixing the white blood cells through stepwise fixation to increase stability thereof, and finally obtaining white blood cell analogs. This patents place emphasis on the selective use of hemolytic agents and unique methods of fixation so that white blood cells could be preserved without addition of lipid substances such as cholesterol. In U.S. Pat. Nos. 6,187,590 and 5,858,790, the methods for preparing white blood cell analogs also involve lysing red blood cells and separating and then fixing white blood cells. The examples of U.S. Pat. No. 6,187,590 to Young also provides a method for preparing five-part differential white blood cell analogs using human white blood cells, with the basic procedures of hemolysis, fixation, washing and preservation as well. Furthermore, U.S. Pat. No. 6,759,246 describes a method of simulating human lymphocytes using porcine granulocytes. Neutrophils and eosinophils can also be simulated by porcine granulocytes, while monocyte analogs can be derived from bovine granulocytes.
The methods in the prior art for simulating human five-part differential white blood cells using mammal white blood cells all entails three basic steps of separating white blood cells through hemolysis, stabilizing or fixing them, and washing and preserving them. These methods suffer from the following disadvantages. The first disadvantage is represented by the complexity of hemolysis processes. For example, repeated use of hemolytic agents for the complete lysis of red blood cells or repeated washing after hemolysis to rinse away residual hemolytic agents are required before separating white blood cells for further fixation. As a matter of fact, during the process of lysing red blood cells completely, the light scattering properties of various types of white blood cells are liable to change, in particular the evident change occurring in scattering characteristics reflective of cell structure complexity of granulocytes. Also, repeated washing not only increases the probability of loss of white blood cells, but also further changes the light scattering property of white blood cells. See FIG. 4. Compared with FIG. 3, it can be seen that granulocytes have disappeared. As is mentioned in U.S. Pat. No. 5,270,208, the fresher whole blood is washed with isotonic salt solutions, its histogram changes over the wash times. Even if it is washed and fixed with solutions containing an aldehyde fixing agent, its histogram is still incorrect, and then addition of a lipoprotein protective agent is needed. The second disadvantage lies in long time of fixation. In order to increase the stability of white blood cells, the fixation time of the prior art is mostly over 2 hours. However, long-time action of the fixing agent will change the light scattering property of white blood cells to various degrees, e.g. increasing the intensity of high-angle light scattering of the cells. The third disadvantage derives from the addition of special components for the preservation solution. As mentioned in above referenced patents, a lipoprotein protective agent must be added in the cell preservation solution. Nonetheless, the addition of special components will increase the costs, and in addition, after whole blood control is formed, lipid substances would influence the stability of red blood cells contained therein and other components are necessary to be added in order to antagonize the side effects thereof.